The present invention relates to isolating, sequencing and cloning of the 5'-flanking region of neuropeptide ff (NPFF) promoter. The characterized neuropeptide ff (NPFF) promoter is useful in gene therapy and in DNA analyses, and in production of gene-modified animals.
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11. A promoter for a neuropeptide ff gene comprising promoter-active DNA fragments located within nucleotide position 1 to position 9840 of seq id NO:1.
4. A promoter for a neuropeptide ff gene comprising promoter-active DNA fragments located within nucleotide position 1 to position 2480 of seq id NO:3.
7. A composition, comprising a coding region operationally coupled to a promoter and an acceptable carrier, wherein said promoter comprises promoter-active DNA fragments which are located in the 5'-flanking region of the human neuropeptide ff gene, wherein said promoter-active DNA comprises seq id NO:3.
1. A promoter for a neuropeptide ff gene comprising a promoter-active DNA fragment located in the 5'-flanking region of the neuropeptide ff gene, wherein said promoter-active DNA comprises nucleic acid selected from the group consisting of seq id NO:1, seq id NO:3, seq id NO:6; seq id NO:7, seq id NO:8, and seq id NO:9.
12. A composition, comprising a coding region operationally coupled to a promoter and an acceptable carrier, wherein said promoter comprises promoter-active DNA fragments which are located in the 5'-flanking region of the mouse neuropeptide ff gene, wherein said promoter-active DNA is selected from the group consisting of bases 9516-9840 of seq id NO:1, seq id NO:6 and seq id NO:7.
2. The promoter of
3. The promoter of
5. The promoter of
6. The promoter of
8. A composition of
9. A composition of
10. A composition of
13. A composition of
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This application is a continuation-in-part of application Ser. No. 09/365,755, filed Aug. 3, 1999, now abandoned, which is incorporated herein by reference.
The present invention relates to sequencing and cloning of the 5'-flanking region of neuropeptide FF (NPFF) promoter. A neuropeptide FF (NPFF) promoter region for mouse, rat and human has been cloned and sequenced. The characterized neuropeptide FF (NPFF) promoter is useful in screening and treating for genetic diseases associated with the promoter area of the NPFF gene by modulation of activation or inhibition of NPFF gene expression through the regulatory sites in the promoter area. The promoter can also be used as a marker for its locus in the corresponding chromosome. Thus, the characterized promoter is of considerable diagnostic value and can be used in gene therapy and in DNA analyses. Further, the promoter can be used for developing genetically modified animals.
Neuropeptide FF was originally identified as a mammalian counterpart of the molluscan cardioactive peptide FMRF-amide (Yang et al., 1985), found in the superficial dorsal horn of the spinal cord, hypothalamus, medulla and pituitary gland (Kivipelto et al. 1989). The findings that the peptide is present in the hypothalamo-pituitary system, decreases during salt-loading and is deficient in the pituitary gland of vasopressin-deficient Brattleboro rats, implicate NPFF involvement in hypothalamic regulation of pituitary functions (Majane and Yang, 1991; Majane and Yang, 1990; Majane et al., 1993). Peripherally administered NPFF raises blood pressure in rats, an effect mediated by both peripheral and central mechanisms (Allard et al., 1995; Laguzi et al., 1996). NPFF has also been implicated in sensory systems, most notably pain and morphine analgesia (Yang et al., 1985). Intracerebroventricular NPFF has been reported to induce a vigorous abstinence syndrome in morphine-tolerant rats. NPFF has attenuated the antinociceptive effects of morphine when administered in the third ventricle, whereas intrathecal NPFF produces long-lasting antinociception (Gouarderes et al., 1993).
The NPFF gene is located in the human chromosome locus 12q13 (Burke et al. 1998), which is known to be associated with a severe condition referred to as Allgrove syndrome (triple-A syndrome). The current NPFF promoter area is an evident region where mutations responsible for triple A syndrome are located. It serves as a useful marker for the appropriate area of chromosome 12, and has diagnostic and therapeutical value in treatment of a triple-A syndrome.
Accordingly, it is an object of this invention to clone and sequence the 5'-flanking region of the NPFF promoter in human, rat and mouse.
The promoter region cloned and characterized here plays an essential role in etiology and/or pathogenesis of CNS disorders involving NPFF, including those associated with deficient regulation of autonomic function, pain conditions, and hormonal dysfunction. Accordingly, another object of the invention is to provide potential methods of screening and treating for genetic diseases associated with the promoter area of the NPFF gene by modulation of activation or inhibition of NPFF gene expression through the regulatory sites in the promoter area. The sequence can also be used as a marker for its locus in the corresponding chromosome. Thus, the characterized promoter is of considerable diagnostic value and can be used in gene therapy and in DNA analyses.
Another object of the invention is to provide genetically modified animals.
FIG. 1A is a schematic representation of the gene structure and cloning strategy for the mouse NPFF gene. Cutting sites for restriction enzymes are indicated by the following abbreviations: B=BamHI, H=HindIII, RI=EcoRI, X=XbaI and Xh=XhoI. The cloning strategy is marked by numbers 1-4. Bar 1 indicates the 4.3 kb EcoRI DNA fragment, bar 2 indicates the 4.5 kb BamHI-EcoRI DNA fragment, bar 3 indicates the 5.5 kb BamHI DNA fragment and bar 4 indicates the 7 kb XbaI DNA fragment.
FIG. 1B shows the nucleotide sequence of the 5'-flanking region of the mouse NPFF gene (SEQ ID NO:6). Sequence is numbered relative to the translational start site (indicated by a bent arrow). The consensus TATA box is shaded. The putative regulatory elements are indicated by arrows underneath the sequences. The AC dinucleotide repeat is indicated by a thick bar under the sequence.
FIG 1C shows: Mouse NPFF promoter sequences. Nucleotide sequence from -9840 bp to -1 bp relative to the translational start site. cDNA sequence is underlined (SEQ ID NO: 1).
FIG 1D shows: Mouse genomic NPFF sequences from coding region and the 3' end of the gene. Nucleotide sequence from +1 relative to the translational start site. cDNA sequence is underlined (SEQ ID: 2).
FIG. 2A shows a map of mouse NPFF promoter deletions fused to luciferase reporter gene. The numbers indicate the nucleotide positions of the 5'-untranslated region with the first nucleotide of the codon for initiation of translation as +1. The promoter fragments were subcloned in NheI-SmaI site of the promotorless luciferase vector pGL3 basic. The MNF5'1.8-LUC reporter construct contains a dinucleotide repeat. Consensus binding sites for transcription factors are indicated by gray-scale colours.
FIG. 2B shows: Basal activity of mouse NPFF promoter to drive luciferase expression. A549 cells were transiently transfected for 48 hours with 5'-promoter deletion constructs fused with a promoterless luciferase vector (pGL3 basic). Transfections were done in duplicate and repeated at least three times. The activity of the promoter was seen to vary with the highest activity in the shortest construct MNF5'0.3-LUC. The activity was seen to drop to almost unmeasurable levels in the MNF5'1.5-LUC construct. The activity was still minimal in the following MNF5'1.8-LUC construct.
FIGS. 2C and 2D show: Activity analysis of the two shortest mouse NPFF promoter constructs MNF5'0.3-LUC and MNF5'0.6-LUC. A549 cells were transiently transfected for 24 hours whereafter the transfectants were stimulated for 16 hours with 10 nM TPA, 10 nM TPA and 1μM ionomycin (IM), 15 μg/ml LPS or 10 μM forskolin. Unstimulated transfectants serve as control. Activity was seen to increase with ∼30% in TPA+IM stimulated MNF5'0.3-LUC transfected cells. A slight increase in activity in the same transfectants was also seen with LPS stimulation. In cells transfected with MNF5'0.6-LUC a ∼50% increase in activity was seen after TPA+IM stimulation. LPS stimulated transfectants also gave an at least ∼40% increase in activity.
FIG. 3 shows a comparison between the first 400 bp of the NPFF promoter from mouse (SEQ ID NO: 7), rat (SEQ ID NO: 8) and human (SEQ ID NO: 9) and a consensus sequence (SEQ ID NO: 10). Sequence similarity is about 90% between mouse and rat and about 70% between rat and human. Conserved consensus binding sites for transcription factors are marked under the sequence. The translational start site is marked by a bent arrow and the TATA-box is marked by a box. M=mouse, R=rat and H=human.
FIG. 4 shows: Nucleotide sequence of the human NPFF promoter. Nucleotide sequence from -2480 bp to -1 bp relative to the translational start site (SEQ ID NO: 3).
FIG. 5 shows: A new, previously unidentified open-reading frame was found in the mouse promoter residing from -4236 to -3841 relative to the translational start site. Amino acids are marked under the sequence (SEQ ID NO: 4). This protein (SEQ ID NO:5) may have multiple functions and it may give rise to a previously unidentified bioactive peptides.
We report here the cloning of the 5'-flanking region of the NPFF gene from mouse, rat and human. Totally 9.8 kb, 1.5 kb and 1.3 kb of the NPFF promoter from mouse, rat and human was cloned and sequenced. Comparisons between the promoter region from all species showed a high sequence similarity. Such a high sequence homology and well-conserved structure could indicate an important physiological function and a need for a similar, tightly regulated transcription of the gene. We have previously shown that the NPFF gene is expressed in specific regions in the brain and in the spinal cord and is induced upon inflammatory stimulus (Vilim et al., 1999). In agreement with this several inflammation related transcription factor consensus sites were found (FIG. 1B), amongst them e.g. NFκB, which is considered as an immediate early mediator of immune and inflammatory responses (Lenardo & Baltimore, 1989). Also several consensus sites for the nuclear factor of activated T-cells (NFAT) were found (for review see Kel et al., 1999). Transcription factors belonging to this growing family play key roles in the regulation of cytokine and other genes during immune response. An interesting finding was also the consensus site in mouse and rat for heat shock factor 1 (HSF1). HSF1 is activated in cells exposed to elevated temperatures and other environmental stress conditions (Sarge et al., 1993). We also focused on the potential involvement of AP1, STAT1 and CREB.
By using transiently transfected cells with deletional series of mouse NPFF promoter fused with firefly luciferase cDNA we could show that the basal activity of the promoter changes with increasing promoter size (FIG. 2B). This indicates that the promoter is tightly regulated is affected by several transcriptional factors and apparently has a complex transcriptional control. To study the possible involvement of some transcription factors our attempt was to a) activate transcription factors by protein kinase C (PKC) b) activate NFATp by PKC and Ca2+ -levels with TPA and ionomycin (Verweij et al., 1990), c) activate transcription factors by the bacterial endotoxin LPS and d) activate CREB by increasing intracellular cAMP values with forskolin. TPA and ionomycin increased promoter activity in MNF5'0.3-LUC transiently transfected cells (FIG. 2C). In this promoter region a consensus binding site for NFAT was also detected by computational analysis (FIG. 2A). It seems likely that this consensus binding site is active and that the transcription factor NFAT contributes to the transcriptional regulation of the NPFF gene. When MNF5'0.6-LUC was transiently transfected in cells and transfectants were stimulated both TPA and ionomycin and LPS stimulated cells showed higher promoter activity compared to control (FIG. 2D). This still confirms the involvement of NFATp and also possibly confirms the function of a NFκB or a STAT1 site residing in this construct (FIG. 2A). An interesting and contradictory finding was that although LPS seems to increase promoter activity, TPA does not. It seems likely that the transcriptional regulation is dependent on a synergistic mechanism involving several transcriptional factors affecting the gene expression.
A possible silencer element was also found in the proximal mouse promoter residing between 1.5 kb to 1.8 kb. Promoter activity dropped dramatically in cells transiently transfected with MNF5'1.5-LUC if compared with cells transfected with the construct MNF5'1.3-LUC, which is only about 200 bp shorter than MNF5'1.5-LUC. A possible silencer could accordingly reside between 1.3 and 1.5 kb of the mouse NPFF promoter. This finding is also confirmed by that cells transiently transfected with the longest construct. MNF5'1.8-LUC, also exhibits a similarly low promoter activity. It would be interesting to see if a longer promoter construct than the ones used in this study could again raise the promoter activity. Since we have cloned and sequenced also the corresponding region in the rat NPFF promoter and the sequence similarity was about 90%, it is possible that this region could be a functional, cell-specific silencer. It still remains unknown if the human NPFF promoter contains similar sequence. The possibility also remains that the AC-dinucleotide repeat in the MNF5'1.8-LUC construct adds additional regulatory effects. Additional roles of the AC-repeat might also be possible since an almost complementary GT-repeat was found approx. 6.5 kb upstream from the TATA-box in the mouse promoter. These partially complementary elements might interact to form complex secondary structures that may in turn contribute to the regulation of this promoter. Similar AC-repeat structures have been characterized in some other brain specific promoters such as PAX-6 gene (Okladnova et al., 1998) and the GLYT-1 gene (Borowsky & Hoffman, 1998). Proteins bound to repeat elements has been reported (Xu & Goodridge, 1998). Thus it is possible to identify this/these proteins bound to the AC-repeat which may play a role in control of the function of the AC-repeat. These proteins might control the accessability of the AC-segment to the complementary GT-repeat.
To study the endogenous NPFF promoter activity in vitro, we tried to affect the transcription of the gene by stimulating a cell-line, A549, which has been found to endogenously express NPFF mRNA. The results are in accordance with the transfection results; the expression of NPFF increased with LPS stimulation, which also was seen in the transfection results. This additionally emphasises the role of NFκ B in the transcriptional regulation of the NPFF gene. The involvement of NFκ B was also confirmed by decreased expression in cells stimulated first with TPA and then with the known NFκ B inhibitor PGA1.
As a conclusion, current data shows that the transcriptional regulation of the NPFF gene is highly complex and probably involves several transcription factors in synergy in control of the gene expression.
It is obvious that key elements involved in regulation of the NPFF gene are included in the characterized promoter, which has not been reported earlier. NPFF is involved in autonomic regulation including blood pressure and heart functions, analgesia and morphine tolerance, learning, and its expression is increased in the spinal cord after peripheral inflammation. Modulation of activation or inhibition of NPFF gene expression through the regulatory sites in the promoter area may be a part of pathophysiology in disease conditions related to these conditions.
The NPFF gene is located in the human chromosome locus 12q13 (Burke et al. 1998), which is known to be associated with a severe condition referred to as Allgrove syndrome (triple-A syndrome) characterized by a triad of adrenocorticotropic hormone (ACTH), resistant adrenal insufficiency, achalasia and alacrima, hypoglycaemia and sensory impairment and autonomic neuropathy. The current NPFF promoter area is an evident region where mutations responsible for triple A syndrome are located. Thus, the characterized promoter serves as a useful marker for the appropriate area of chromosome 12. Also in other applications it is of considerable diagnostic value and can be used in gene therapy and in DNA analyses.
The invention will be further described with reference to the following non-limiting examples.
Cloning of the Mouse NPFF Promoter
Stratagene mouse genomic library was screened with mouse NPFF cDNA (Vilim et al., 1999). DNA from positive clones were isolated, digested with a panel of restriction enzymes and analysed by Southern blotting. A 4.3 kb EcoRI DNA fragment (see FIG. 1A) that hybridized with the mouse NPFF cDNA probe was subcloned in pBluescript KS+/- vector (Stratagene). The cloned 4.3 kb EcoRI fragment was sequenced by automated sequencer (ABI Prism Automated Fluorescence Sequencer) by using universal primers (T3, T7, Promega) and gene specific primers. Restriction enzyme map of the resulting sequence was computed by using the University of Wisconsin GCG Sequence Analysis Software Package. A 0.8 kb BamHI-EcoRI fragment at the 3'-end of the 4.3 kb EcoRI fragment was subsequently used to probe the Southern blot of NPFF lambda-DNA. This probe hybridized to a 4.4 kb fragment of BamHI-EcoRI digested NPFF lambda-DNA. The 4.4 kb BamHI-EcoRI fragment was thereafter subcloned in pBluescript KS+/- and sequenced as described previously. A 3.5 kb EcoRI-BamHI fragment located at the 5' end of the 4.3 kb EcoRI fragment was then used to reprobe the NPFF Southern. This probe hybrized to a 5.5 kb BamHI fragment, which in turn, was subcloned in pBluescript KS+/- and sequenced. A 2.5 kb BamHI-XbaI fragment at the 5'-end of the 5.5 kb BamHI fragment was finally used to reprobe the NPFF Souther filter and it hybridized to a 7 kb XbaI fragment. The 7 kb XbaI fragment was subsequently subcloned in pBluescript KS+/- and sequenced. The resulting nucleotide sequences were assembled using the GCG Sequence Analysis Software Package. Total of 10.3 kb of the NPFF promoter was cloned and sequenced. The search for the consensus transcription factor recognition sites was performed by MatInspector Professional from Transfac Database at NCBI/NIH.
By using the strategies presented in examples 1 and 4 a 9.8 kb, 1.5 kb and 1.3 kb piece of the NPFF promoter region was cloned and sequenced from mouse, rat and human, respectively. An illustration of the cloning strategy of the mouse NPFF promoter is shown in FIG. 1A. The homology between the proximal promoter based on sequence similarity was seen to be high between all species when doing comparisons based on computorial software (GCG, Wisconsin/CSC, see FIG. 1B). Some of the consensus binding sites for transcription factors were seen to be conserved between all three species in the proximal promoter (FIG. 3).
Luciferase Reporter Constructs
To prepare 5'-deletion constructs of the proximal mouse promoter a 325 bp, 579 bp, 805 bp, 1085 bp, 1289 bp, 1527 bp and a 1861 bp fragment were amplified by PCR with the antisense primer 5'-TGG AGT CCA TGC TGC CAT-3' (SEQ ID NO: 11) and the sense primers (cutting site for NheI is underlined) 5'-GTG CTA GCA ATC TGT TGA AGG ATT GG-3' (SEQ ID NO: 12), 5'-GTG CTA GCA GTC TCC TAT CTC TCA CT-3' (SEQ ID NO: 13), 5'-GTG CTA GCA GAC GGA ACT GGA AAA AT-3' (SEQ ID NO: 14), 5'-GTG CTA GCT CTC CTA GCA AGT AAT TC-3' (SEQ ID NO: 15), 5'-GTG CTA GCT ACA TAT GAC TGA GAG AT-3' (SEQ ID NO: 16), 5'-GTG CTA GCA GCC TGG ATG CAT TGT AT-3' (SEQ ID NO: 17) and 5'-GTG CTA GCA CAG AGT CTC AGG CTT AG-3' (SEQ ID NO: 18), respectively. PCR reactions were performed with Pfu DNA polymerase (Promega) using an Eppendorf Mastercycler gradient machine with the following program: 1. 95°C for 2 min, 2. 95°C for 45 sek, 3. 53.6°C for 30 sek, 4. 72°C for 3 min with a 1 sek addition after each cycle, 5. Steps 2-4 for 35 cycles followed by a 72°C 5 min final extension. The PCR products were cleaned from primers and nucleotides with QIAquick PCR purification kit (Qiagen) and subsequently cut with NheI. The cut products were run on a 1.2% agarose gel, excised from the gel and extracted by QIAquick agarose gel extraction kit. The PCR products were ligated in NheI-SmaI cut promoterless pGL3-basic vector (Promega) upstream of the firefly luciferase cDNA in sense orientation. The yielded constructs MNF5'0.3-LUC, MNF5'0.6-LUC, MNF5'0.8-LUC, MNF5'1.0-LUC, MNF5'1.3-LUC, MNF5'1.5-LUC and MNF5'1.8LUC (see FIG. 2A) were transformed in competent DH5α cells.
Transfection and Luciferase Assay
The functioning of the promoter in a cell was shown.
The human lung carcinoma A549 cell-line was kept in a humidified cell-incubator at 37°C and with 5% CO2. The cell-line was grown in Dulbecco's modified eagle medium (Gibco) containing 10% fetal calf serum (Gibco), 1×Glutamax (Gibco), 50 μg/ml penicillin and 50 IU/ml streptomycin (Gibco) and was regularly passed. 16-24 hours before transfection cells were plated at a density of 1×105 cells in 1 ml medium in 12-well dishes (Nunc). Reporter constructs (1 μg) or promoterless pGL3-basic vector (1 μg, as negative control) were co-transfected with pSV-β-galactosidase vector (0.5 μg, Promega) by first mixing 3 μl of Fugene 6 (Roche) transfection reagent with 57 μl of serum-free medium, which was incubated at RT for 5 minutes. The mix was added to the DNA and incubated at RT for 15 minutes and then added to the cells. The transfections were done in duplicates and repeated at least three times. Cells were transiently transfected for a total time of 48 h and before collecting the transfectants some cells were left unstimulated or stimulated for 16 hours with TPA (10 nM), TPA (10 nM) and ionomycin (1 μM), lipopolysaccharide (LPS, 15 μg/ml) or forskolin (10 μM). Cells were collected and lysed with 140 μl of reporter lysis buffer (Promega) according to the manufacturer's manual. The luciferase assay was performed on 20 μl of cleared cell extract and 100 μl of luciferase assay reagent (Promega) using a Luminoscan luminometer (Labsystems). Transfection efficiencies were determined by using β-Galactosidase enzyme assay system (Promega) with 50 μl of cell extract. The luciferase acitivity of each transfection was expressed as luciferase activity/β-galactosidase activity.
The ability of a deletional series of the NPFF promoter to drive the transcription of firefly luciferase cDNA is presented in FIG. 2B. The highest expression of luciferase was seen in the shortest construct (MNF5'0.3-LUC) with decreasing activity in the two following constructs (MNF5'0.6-LUC and MNF5'0.8-LUC, respectively). The luciferase expression started increasing again with the two following constructs (MNF5'1.0-LUC and MNF5'1.3-LUC, respectively) whereafter the expression decreased to barely measurable level with the last two constructs (MNF5'1.5-LUC and MNF5'1.8-LUC). The last construct also contained a 29×(AC) dinucleotide repeat.
To study the involvement of some known pro-inflammatory transcriptional factors the two shortest constructs (MNF5'0.3-LUC and MNF5'0.6-LUC) were transfected in cells and stimulated with a phorbol ester, a phorbol ester together with a Ca2+ -ionophore, a bacteria endotoxin and a cAMP activator. These compounds should all affect several transcription factors, amongst them many known pro-inflammatory transcription factors eg. NFκ B, NFAT and CREB. The results are presented in FIGS. 2C and 2D. The results show an increase in luciferase activity of TPA and ionomycin stimulated MNF5'0.3-LUC transfected cells (FIGS. 2C and D) and an increase in luciferase activity in TPA and ionomycin and LPS stimulated MNF50.6'-LUC transfected cells.
Cloning of the 5'-flanking Region of the Human and Rat NPFF Gene Promoter
To clone the promoter region of the human and rat NPFF gene, a Genome Walker kit (Clontech, Palo Alto, Calif.) was used according to manufacturer's instructions. The NPFF specific human and rat antisense primers used for the primary PCR reaction was 5'-GCT GCC ACC ACC TAC CCT CCT AC-3' (SEQ ID NO: 19) and 5'-CAC CCC AGC TCC CTG CCT CTT-3' (SEQ ID NO: 20), respectively. The antisense primer for the nested human and rat PCR was 5'-GTG GAT CCA TCT AGA GCA GGC AAA TG-3' (SEQ ID NO: 21) and 5'-CGT GGC CCC AGT TCC TCA GCA-3' (SEQ ID NO: 22), respectively. The PCR reactions were performed using a MJ Research MiniCycler. The primary PCR reactions was performed with the following conditions: 94°C for 25 sec and 72°C for 4 min×7 cycles; 94°C for 25 sec and 67°C for 4 min×32 cycles, followed by a 67° C. 4 min final extension. The nested PCR reactions was conducted using a 50 times diluted primary PCR product as a template, the same reaction composition and cycle parameters, except that nested primers were used and that 20 thermocycles were performed instead of 32. Using this technique, we were able to identify a single major PCR product in one of the human libraries (PvuII) and two major PCR products in the rat libraries (DraI and PvuII) provided in the Genome Walker kit. The products were purified with the QIAquick PCR purification kit (Qiagen) and cloned into pGEM T-vector (Promega) and sequenced automatically in both directions (ABI Prism Automated Fluorescence Sequencer). Using this approach 1.3 kb of the human NPFF promoter and 1.5 kb of the rat NPFF promoter was cloned and sequenced. We still continued our cloning efforts conserning the human NPFF promoter and by using a second round of the same approach as above we were able to clone an additional 4 kb fragment of the human promoter. Sequencing is now in progress. A comparison between the NPFF promoters from all species is presented in FIG. 3. The search for the consensus transcription factor recognition sites was performed by MatInspector and MatInspector Professional from Transfac Database at NCBI/NIH. Comparisonal studies to study homology between the NPFF promoters were performed using Seqweb software package available at CSC web site (www.csc.fi).
The human sequences have been presented in FIG. 4.
Comparison of Proximal Promoter of NPFF Gene Between Mouse, Rat and Human
A comparison between the first 400 bp of the NPFF promoter from mouse, rat and human. Sequence similarity is about 90% between mouse and rat and about 70% between rat and human. Conserved consensus binding sites for transcription factors are marked under the sequence. The translational start site is marked by a bent arrow and the TATA-box is marked by a box (FIG. 3). M=mouse, R=rat and H=human.
The invention has been illustrated by examples and embodiments, but it may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention and all such modifications are intended to be included within the scope of the enclosed claims.
References
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Burke, J. F., Clarke, A. J. H., James, M. R. & Perry, S. J. (1998). Physical mapping of the human NPFF gene and investigation of its candidacy as a disease gene locus. Soc. Neurosci. Abstr. 24(2): 2046.
Gouarderes, C., Sutak, M., Zajac, J. M. & Jhamandas, K. (1993). Antinociceptive effects of intrathecally administered F8Famide and FMRFamide in the rat. Eur. J. Pharmacol. 237:73-81.
Kel, A., Kel-Margoulis, O., Babenko, V. & Wingender, E. (1999). Recognition of NFATp/AP-1 composite elements within genes induced upon the activation of immune cells. J. Mol. Biol. 288: 353-376.
Kivipelto, L., Majane, E. A., Yang, H. Y. & Panula, P. (1989). Immunohistochemical distribution and partial characterization of FLFQPQRFamidelike peptides in the rat central nervous system of rats. J. Comp. Neurol. 286: 269-287.
Laguzzi, R., Nosjean, A., Mazarguil, H. & Allard, M. (1996). Cardiovascular effects induced by the stimulation of neuropeptide FF receptors in the drosal vagal complex: an autoradiographic and pharmacological study in the rat. Brain Res. 711: 193-202.
Lenardo, M. J. & Baltimore, D. (1989). NF-kappa B: a pleiotropic mediator of inducible and tissue-specific gene control. Cell 58(2): 227-229.
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Majane E. A., Zhu, J., Aarnisalo, A. A., Panula, P. & Yang, H. Y. (1993). Origin of neurohypophyseal neuropeptide-FF (FLFQPQRF-NH2). Endocrinology 133: 1578-1584.
Okladnova, O., Syagailo, Y. V., Tranitz, M., Stober, G., Rieder, P., Mossner, R. & Lesch, K.-P. (1998). A promoter-associated polymorphic repeat modulates PAX-6 expression in human brain. Biochem. Biophys. Res. Commun. 248: 402-405.
Sarge K. D., Murphy, S. P. & Morimoto, R. I. (1993). Activation of heat shock gene transcription by heat shock factor 1 involves oligomerization, acquisition of DNA-binding activity, and nuclear localization and can occur in the absence of stress. Mol. Cell Biol. 13(3): 1329-13407.
Verweij, C. L., Guidos, C. & Crabtree, G. R. (1990). Cell type specificity and activation requirements for NF-AT-1 transcriptional activity determined by a new method using transgenic mice to assay transcriptional activity if an individual nuclear factor. J. Biol. Chem. 265: 15788-15795.
Vilim, F. S., Aarnisalo, A. A., Nieminen, M.-L., Lintunen, M., Karlstedt, K., Kontinen, V. K., Kalso, E., States, B., Panula, P. & Ziff, E. (1999). Gene for pain modulatory neuropeptide NPFF: induction in spinal cord by noxious stimuli. Mol. Pharmacol. 55: 804-811.
Yang, H. Y. T., Fratta, W., Majane, E. A. & Costa, E. (1985). Isolation, sequencing, synthesis, and pharmacological characterization of two brain neuropeptides that modulate the action of morphine. Proc. Natl. Acad. Sci. USA 82: 7757-7761.
| SEQUENCE LISTING |
| <100> GENERAL INFORMATION: |
| <160> NUMBER OF SEQ ID NOS: 22 |
| <200> SEQUENCE CHARACTERISTICS: |
| <210> SEQ ID NO 1 |
| <211> LENGTH: 9840 |
| <212> TYPE: DNA |
| <213> ORGANISM: Mouse |
| <400> SEQUENCE: 1 |
| tctagacaga taccaggtac caatgttaaa tcatgatctt acgttttgtg tcagaactta 60 |
| ttttgcatgc cattcatgac tctggagagt tttctccaaa aggaaagatg agattaattt 120 |
| tcttttgcat tctttgtaat tatgacaaag aagagtcctt tctgttcctc tgtcctattt 180 |
| tctaaccact tgctctctac tgtttgccct ccttcctggc agaaagtccc aaggaaagct 240 |
| cagagccaac gggttctcca gccccagtga ttcagcacag ttccgcatca gcccctagca 300 |
| atggccttag tgttcgatct gcagctgaag ctgtggccac ctcggtcctc actcagatgg 360 |
| ccagccaaag gacagaactg agcatgccca tacagtcaca tgtgatcatg accccacagt 420 |
| cgcagtctgc gggcagatga tgcctcccat gatggagagg tccccagcca gagctcgccc 480 |
| gcctgagcca agagagagat caacttaacc cctcctgtct gtcttggaat tgggcagaat 540 |
| ggagagggag aaattgtgtg ttgtgagtat ggacagatgt ggggcttttc tctttagcca 600 |
| catgatcatg tacgattaac acctgtacta ggcgcttctg gccccaaagc cacatagatc 660 |
| atcccccaag ggtagggttt cttatgtacc tacagccaaa ggcctttcca tatggtctga 720 |
| gcagtcatcc ctggccctga tgcatgtgtg cctgtctctt aacactaacc cttgcacttg 780 |
| taggtttggg gtttctcagt gtcccctcct cccattgacc tgtggctgtt accttcttat 840 |
| ttcttattag catgccactt cctgccagat ggagggaggt gagatttgat ggcgtgttac 900 |
| ttgcttgtcc cacccctagc caccacatgc actcaggact ttgtctccca atagctgctt 960 |
| atttgtctct ttcctccttc ctaaactgca gtgaaaacat tcacttgttg agaatgtaat 1020 |
| acatttaatg tatgaggtag cagtgtttcc cacctcttcc atgtgcttta ttcctatctg 1080 |
| ctaccaaaaa caaacacaaa caaaaaaagc aaaactctga gagtttgaat catttttcat 1140 |
| ttccaaatct attggtacct catttcgtct cttgactttc cttagtctag tggtggggtc 1200 |
| tttcctactt ccccactagg ccttgggaac ctcatcttgt ggccttattg ttagtggcaa 1260 |
| tgaaaaagga gagagctgga gacctaactg ggcttccctg tctccttccc taccctccag 1320 |
| ttctaatcat cagggcagac aggaacagtg taatttaaaa cttgttccat caggttactg 1380 |
| gattaactga ttcttttatg ttttacaaga gtttactggc caaagtctac atattgtcat 1440 |
| ctttggtcat ctgtgctcct gcctctccac tgtgcttccc tgtccacatc actgcctgtt 1500 |
| ccactataac tactttagct cagacctcct agagttgtca gacagtagct gcttccattc 1560 |
| tctgtccctc ctacagctct cagcctccct tcttttctaa ggtatgtttt gtacccacca 1620 |
| gtgtccagca ctctctcaac aaccttttca tcttctcacc cagttcttag cctacccact 1680 |
| ggttgtctct agtcctgaaa tttcgttgaa cttgtccatg gtacaggctc cagggctctg 1740 |
| aagcatcatg gttgaggtca tggatgcttt tgaatataag actgagtgga gagaggaggt 1800 |
| atacattttc ctatgtataa tctcagcaga gcactcagga tatggcctgt ctgtgagatt 1860 |
| gctgtctgtg gctgttggct gtccttattt tatgggttac agagagaaaa tacacccctc 1920 |
| ctcttcttca tcttcccttg gctctgagct atgcaggctc ttccagagcc agaggcctgt 1980 |
| ggatagatag gtcagcttta atagctcttg agttgagaat ccttcccatt gtcctagaac 2040 |
| caccttctgc catctgctca agccaagtcc tcttttctag ctccatctta agtatgcaga 2100 |
| ataattgctt gcgtctttgt cttccaaaat tcactgtcgt ggaggaatgg aaatgtcgtg 2160 |
| tagcttatat gattatttcc tcagagttta gactagtgag cccatcctgt gacatgtttg 2220 |
| tatgttttat gtaaatttcc ctcctgctct ttagagtcaa tgctgaacag gccacaccca 2280 |
| gtgaaaacta ggaactggtt ttatagtctt ctcccacagg gtcttaacaa aaacatcccc 2340 |
| tgaggtgaca aggatagcaa atgccacagc agatggttga gggcaagcca ccatctccag 2400 |
| gggtttcact tggccttaga aactcacagc catagtttga gctcaggact tctttagatg 2460 |
| gctgcttcct aggatttttt tttcctgctt atgaattttg tttctttttt tttaattgtc 2520 |
| ttgatttccc agtagcagcc ttacactaaa atatgactga gcttatagct tccaagggcc 2580 |
| ccccttggct attttcttcc tccatcagtc aagtgtttaa ttcagtgtaa cctaccagtc 2640 |
| tgtcctggtt gcatgtctag tatacgtgga ggttcttttt cactttcttg acccttcatg 2700 |
| tctgcttctc ttgagtcttt gtttttatag caggaagtta gtattggggg cttgaatgat 2760 |
| gcagggcacc aacagaacca ttgcaggact gaaatcccca gactaccgat accttggtgg 2820 |
| tcggttctca gcttcactaa gaaagcagaa cggctgctta tgctgaagcc tctgtgacag 2880 |
| tcaagggggt catcacctac attattgctg ccaggggtca cagccctgac ctttgccttc 2940 |
| cagacttaac tgaaccagaa ccagatacca tagaggatag caataaaccc ttcttgacat 3000 |
| ctgactatga tgtgtcatag cggggtctct ggtcatgtct atttggggtt caatatgcct 3060 |
| cttgtatttg aatgtcaact cctgtcccta ggtataggaa attttgtgat ataatttcat 3120 |
| taaatagttt gactaattcc tttttaattt atctcaggtc ctccttctcc ccagtggctt 3180 |
| ctgaggtttg tttggtcttt taagtatatt ccaagcttct tgaaaatact ggtaatatct 3240 |
| atctctctct ttctctctct ctctctctgt ctctgtgtgt gtgtgtgtct gtctgtctgt 3300 |
| ctgtctgtct gtctgtgtgt gtctgagtat atgattgttc caactttgtg ctccactgca 3360 |
| gaactgcatg atcttaatct cttggggaca ctttctagtg agggttttca ctttcatgtt 3420 |
| tctttcatgt taatttctat cttgttcctt ttccatgttt gaatgccctc atcaaaatct 3480 |
| ccctccatat atctgacttt ctcctccaac ttacccactg ttttgttcat tttggaggtt 3540 |
| ttgtatacag ttcctagttc atgcatttgt atctgagact tctacatcaa ctctgaactc 3600 |
| tttctcagac ttctcagtac ttttggtttt cattaaggag aggtttgtag tatgccctgg 3660 |
| tctgctgttg tctcatgttc cttgtgtttc tctgtggaat tttgtacatc agtcaggatg 3720 |
| gctatgtcct ccagttttaa ctcatctttt cccaactctg ttttgtttca tgtaactaag 3780 |
| cccttcttca gaggtctgtc agaaaggcgc tgagagtgtg atgtctcagg cccaggtgtg 3840 |
| gtcagaggcc tggtctgtgc tcttgccacc cactccatat aacacagagt gttcagagtc 3900 |
| tcaaaggtga gaagccgccc aacacgcagg gagagctggg aaggacacgg gccccttcat 3960 |
| tcactttcac tcccttcctg ttggctgaga tgccagatct gcctgcttcc ttcctccact 4020 |
| ccttcccatg gctgtaagca ccagaccaca tacttaaggg agctgtgctg gtgaaccttg 4080 |
| ccttgctgcc tgccttccct ctcccattct gtagctcttg actccttcct tgattcacct 4140 |
| cttccgtctc ctcagggacc cgccctccag caccatcatc ctgagagata taactgtact 4200 |
| ttgtacagcc tgaaccgcca aaaagacaca catgcaattc ttcctctggc ttctgagagg 4260 |
| ctccttaaag gtgctaactg ctcagctcac tctccccggc gtgtccccat cctcagaaca 4320 |
| catttccatt atctattgta cccaccaaaa agaaatatgt acttcttatg aaaagaaaac 4380 |
| cctagtctgt tcagatgtgt ctcacagctg tgtgacacgt gccttcgttg ctatgctttc 4440 |
| tcctttcttt agccatgttt gaccagggtg ggagggtgga tcctaaagcc tatcaaaaga 4500 |
| ccctacccca ctccagtcca gctagacatt cttcttacaa attctgtttc tgtctgtata 4560 |
| tgtgcatatg catagacaaa tccccctatt ccaccagcct ggtgatccat aggaatgagc 4620 |
| agtgcctgct ggccacattc ccaccgtttg cactgttact ttgaggtaaa atcctaccct 4680 |
| agaatgaaca aaggctggtg aaagtagggc agattaaggc agcttatgtt cttgtaaatg 4740 |
| caagtttcta tttcagctag taggtgtttt ctttttctgt tttgtttttt aatgtagagc 4800 |
| tggtaggcta taagccagca attgtgagta aggctttagc tattagtggc tgtgagcact 4860 |
| agtttcattg actttacctt agggcagtgg ttctcagaac atggtccaag aacaacatca 4920 |
| gcagcaccat cacctgaaga gcttgctaga aatgtacact ctgggccatc ccaacctcct 4980 |
| gagttggcca ctctgaaggt gagctttaac taacagtctc tgctgctagt tcacactaat 5040 |
| gcatgacagt ccctagcgga caggctggga gcatcttagc tctgggatga caacgattac 5100 |
| tttaaatgtc ttctctgcct tagaattgat atttttattt cccccagtcc ttccttcctc 5160 |
| ttccattaaa acagccacca ccacattact catctcaaat tctaggttgg tcttccttct 5220 |
| agtcttagct ctaaaactct tgcctgcatt ggtctggact catatttcct tgcaggctac 5280 |
| tagttctgca ttcttggtga ctttagccag caggaaggcc aggagtggtg gtggcatatg 5340 |
| cctttgatcc cagcactttt gagggaagca gaggcaggca gatctctttg tgttcaagac 5400 |
| cagtctggtc tacataggga gttcaaagcc aatcatagct atgcagtgag accctgtctc 5460 |
| aaaacaaaca aacaaaatca gcaggagcct tagttgtcca tttcttccct gtgcacacac 5520 |
| cacatctctt acaggaagat tagcctccac ccccacagtg gagcctccta catcctgata 5580 |
| gagtatatgt tgagaagcca tgtgtatcta tgaatatagc tctgttctat atccttttga 5640 |
| catgtagcaa tacctctcca tcctcaagga actcaaccca gtctgggtct ccccaggctc 5700 |
| cagtggtaga ctctgacagg tgggaggata cagtgctctg ggctgttttg ttacaaaagt 5760 |
| gtcttctgtc ctttccctcc tcccaattca gcatgacccc tgtgagcagg ctctcacaat 5820 |
| ctcctggggc agggctgagg caggggcttt cagctcttct ccataactat cccttcttcc 5880 |
| ttcccccatg ccatttagca gttatcaccc agccttgcct tctccctcca tcccttgccc 5940 |
| tgacatatac tgtgccttat ttatgctgca aatataacat taaactatca agagaatgac 6000 |
| tggtatgttt ggtgcttccc tacgcagact catggggccc attggtcact cctagagact 6060 |
| cagtaggcat ttgtgtctga ccatcctcct ccttccactt cttagggcag aactagcagg 6120 |
| ctctctctgc tttcagtaag taacatggtg ttggaaaagg cacagagttc agatctttaa 6180 |
| actgcctcag agccaaggca tcacaaaaag actgaccaat gggaatactg aacaccctgg 6240 |
| ctctttcagt gttttatgct cacccacttc caacaattga aaggaagaaa aagtcctact 6300 |
| cccaagaaag gggcttggga gtgtacaaag aggtagacaa agtcaagctt tctccagaga 6360 |
| ctagaaggaa tagctgaaga gatggctcag tagttaagag gaaaaactgc tcttgtctca 6420 |
| aaagatccgt ttggtcccca cacccatgtt ggctagttta ccaccaccct taactccagc 6480 |
| tgcaagggat ctggtgccct cttttggccc ccacaggcac tgcactcact tgcataaccc 6540 |
| ttcccccagc acacatatac acaattaaaa agttaaaaaa aaaaaaatta aaaagagaag 6600 |
| aagattggaa actcgaggca aactttgtaa aagcagatta aagctcacag gagaacaggt 6660 |
| aatgatcagg gtgaggaagc ggacaggtga gccactgatc ctttctgtgt ctgtgtcttc 6720 |
| cactaaaagt ggaaaccacc aaggagacag actgaagaac ctgacaaaag acagaacagg 6780 |
| tacctctaag gttccttggt agaacagatc tactgggttg gtgtctggtg aaggactcag 6840 |
| agcctcctta ggaaatggaa acacttactg gccatcactg tgtgggcccc agcaattaag 6900 |
| gtacttactg ccaagcctga agacctgagc ttgatccctg gatacatgtg gtgggaagag 6960 |
| aactggcaag gtgttctctg agctccacat atgctctgtg gggcatgtgt ctccttctcc 7020 |
| ccaggtaaat aaatgaatga gaaagtgggt ggggagcaca cagtatgtcc aagaaagaga 7080 |
| gaacattacc aaaagctaag acagagtctg gaggaagact ggagaggtgg ctcagtggtt 7140 |
| aagagcactt gtgttctaga ggacttgagt tccgttccat ttaggtggct cacatctgga 7200 |
| attctggact ttcggaagaa cagtcaggtg ctcttaccca ctgagccatc tcaccagccc 7260 |
| caatattttt tttttttttt tttgagacag ggtttctctg tgtagctctc actgtcctgg 7320 |
| aactcattct gtagaccagg ctggccttga actcagagat ttgccctacc tcccaagtgc 7380 |
| tgggactaaa ggcatatgtc accacaggcc agctgagatc ctgtatttaa ataaataagt 7440 |
| ctggaaggtg ataaataaaa ctaagtctag aagatgagaa tcctagcaca caggttagga 7500 |
| tgattaattt ttgttgaggt tagaagtgaa ccagcttctt tgtgaactta gtagcttcag 7560 |
| cccagactcc ggtactgaag cagcagtgca gtgaacacag ggtggctgct gtgagactgc 7620 |
| tgtgtacgca acccatctgc tgttcaggac agcttcctgt tcacagggtt aggttttttt 7680 |
| attgttctct gggtgctgga gattgtgctc aggggcctcc agcaagttgc ctctttgttg 7740 |
| tttttttgtt tgtttgtttt gttttgaggc agggtttctc tgtgtagcct tggctgtttt 7800 |
| ggaactagct ctgtagaaga gcctggcctc gactcaaaaa gatcagcctg cctctgaagt 7860 |
| cacacagtga atttcgagag caaagataaa atacgaaatt tctgccaggt gtggtggtgc 7920 |
| acatctgtaa tcccagcact tgagaaacta aaaacagagg ccagcatatt gggctacata 7980 |
| cagagtctca ggcttagaaa accagacaag ctgggcactg tgacacacac tttaatccca 8040 |
| gcacttggga ggcagaggca tcctaatcta catagtaaat tctagaccag ccaaggctaa 8100 |
| atggtgacac ctgtttacac acacacacac acacacacac acacacacac acacacacac 8160 |
| acacacacac acacagtgga gacaggaaaa gagagaggtg gggagagaaa ccgagaagac 8220 |
| ccacaacaaa agcagcagtg aaatatttca actataatgt atgacaagct ctacaggaaa 8280 |
| tcttgagaca aaccttacag aaaggttcac ccaagcctgg atgcattgta ttctagaaca 8340 |
| tcagaaacct gatctagaag gtctccctct gcagtagagc acctgcctac catacacaag 8400 |
| gctcttcatt tggacttgat tcccaagaaa gaaattttaa aatgccactt atccgcaaac 8460 |
| ctaaaatgta agttggttcc aataaagtac ttccctgttg agcaaagaag atgtggatac 8520 |
| atggtatttc tccaaatatg tgaggggctt ttacatatga ctgagagata acctgagact 8580 |
| ccattgaaca agctaggacc tctggtagag gccaccaaga agctaaagcc cactcagcac 8640 |
| tctctggaga tggtaagttc cccaggactg gagtgggagg acagcaaagg gaatcacagt 8700 |
| tgacattttg aaaacaccgg gtctgtgctt tcctacaaaa tgcatcccaa atgtttctcc 8760 |
| tagcaagtaa ttcattttac tgttcccata tgtaagtgag gaaaaaaaga gtgtgagcag 8820 |
| cttgctttgc tcatggggta gaaccccgac agtccttctt ctgtttaggc tagagacatg 8880 |
| gtactctgac acctggattt gcaagtgagg ttaggactag ctcctttaaa ggacccttcc 8940 |
| ctgaactgga gtgattgtct gtccctaaag cagaacccta gtcgccagct ccagtagtat 9000 |
| attagaacca gaaccaggca gagcccatgc tgaccagacg gaactggaaa aatgtcacaa 9060 |
| ttctgggccc caaagaacta ggtcctcaag tcctagacaa aatgtatgga aagggaaatg 9120 |
| gctggacgtg gcagtgaaga gtagtggcca caaggtggca gcagagtttc agctgtggag 9180 |
| gcccaatccc cagttctctt gcaaagatgg gcctgtccac aaaatttaca ggccacctct 9240 |
| actcagtaag gctccaaaaa gagtctccta tctctcactt aactattcac aggtaaatct 9300 |
| taaagggtag tgaacccaca tttaacctga ctagaagcag tggggattga aatggggctg 9360 |
| tggtcctgat cacccattcc aggcaggagt agggaccaag ctggttcacc ctagcctgca 9420 |
| cttaacacta gttccttccc atccaggaca taatgcccaa ttctgacagg agtttctcca 9480 |
| gtcaggaaca agaggtgatc aattgaagct tctccaatct gttgaaggat tggaggttct 9540 |
| tctaaggttc ccccagggtc taactctgac aaactgtctg caattaatga tgcttcctga 9600 |
| gctccggaga caagatttat gcatctaata aagtctatat aactccaggc ttaggctggg 9660 |
| ggggaggaag ctaagagcag agagtcccca ggggagtacg ggaggggggg tcccaggtgg 9720 |
| ctcttaatag agccatgcat ttccattgct tgtctagatt tccccccagg ctgccggtga 9780 |
| ggtgggggta gggacatcag gtataagaag accgtgggca ctcaggaggc agatggcagc 9840 |
| <200> SEQUENCE CHARACTERISTICS: |
| <210> SEQ ID NO 2 |
| <211> LENGTH: 5124 |
| <212> TYPE: DNA |
| <213> ORGANISM: Mouse |
| <400> SEQUENCE: 2 |
| atggactcca agtgggctgc tctgctgctg ctgctactgc tgctgctgaa ttggggccac 60 |
| actgaagagg cagggagctg gggtgaagac caagtctttg cagtgagtga acatcctgct 120 |
| gccccatcca cccagccgcc atctctcctt cagctgaaac ttatgataca gggtttggtg 180 |
| ttccccaggc tgctaaggtc ttcaaaaagt ctggcttcta agggagggaa atggcttctc 240 |
| agcccacgca agtttcccct agaagccatt ctttccctcc atgtcttcag tgtacaagac 300 |
| actgccaggt tccttccctg ccactctctc atcacaggga gaagataagg gaccccaccc 360 |
| accacagtat gcccacattc cagacaggat ccagactcct gggtccctct ttcgtgttct 420 |
| gctccaggcc atggacacac ctagaaggag cccagccttc ctgtttcagc cccagaggtg 480 |
| agttccaaag ggaagaggct gagaagggtg gagggcagag gaagatgtga gagaaggtga 540 |
| gatggaggga gtgccaacta aggatgaatc tctcctaagg tttggcagaa gtgcatgggg 600 |
| gtcctggagc aaggaacagc taaatccgca ggccagacag ttctggagcc tggccgctcc 660 |
| tcagcgcttt gggaagaagt agcatcgtca gctgtgatgc ctgcatgcaa aaccacttcc 720 |
| ccatgttccc tgtgtgcccc caaataaaaa tggtccggct ggcttcagaa tccctgtgtt 780 |
| tggacaagac tgtcagggag caggtgggag cccaagggca atagctgtag ccccccttca 840 |
| cctccactca gtctctagcc attctgttgt taaggatccc caaggctact actgcacctt 900 |
| gcctcctctc ggtaacaaaa aagaacaagg ggttcaaaag gagaacaagc tcaccatgtt 960 |
| tattccttat accctcatga cccaaggcca gagagagcag ggttttggaa gccaaagagc 1020 |
| agcatttatt caggactcca atagattcat ccatcaccca cggaatgagg acaaatcctg 1080 |
| tgctggctgg ggccctgtgg ttcatggctc cttgcttgcc tgtgccttcc tcagtctcaa 1140 |
| ggcagacagg ctgtgtcaga ggtagagatg gcacttctgg agggtaccag agctaggtgg 1200 |
| atacatggac ccaggggcag aaggagcaag aagtaaaaga tgcatatcca tcactgcagt 1260 |
| gggatgctac ttgctacccg ccatgatcct gaggtactgt agggcgcggt gagcagcatc 1320 |
| acctcgggct gcctccctgg tggttgcaga accataacac acagtggctg gctgggtgga 1380 |
| cagttccact aggcactggc agagcccact caggctcagt tcctctggaa accaagaaag 1440 |
| agaagggagg cactggtggg gaaggggcac caaacagtgt acacatcacc catctcccaa 1500 |
| ctccttcata cttgctttga tccagtcccc tccatcccca gtctctagtt ggccatacca 1560 |
| atatccagat agctgacatg gaaagcctgc tcctcagaga gctcactgag gacactgcag 1620 |
| caggcagagc ccagagcccc tagagagccc acggagcaac tgcgaaggga taggatcttt 1680 |
| tctcccacag aattccgcaa ggaatcccag gtgcagcctg gcccacgatt cctcagtcca 1740 |
| tccaggcggg agctcacgcc ctaatgagat aagagtgaga aagcagctaa ggactgtggg 1800 |
| acccaagaac tgggtattat gctctgactc ctggcagcct tcctcatccc aggaaaagtt 1860 |
| gcctataggg gctttgccct aggccagctt tcactagtga ccctacatat aggctgaggg 1920 |
| gacttctggg cgcacaggtt caaggagccc tgggaaggaa agcaggccca tactcacttc 1980 |
| caaaccacag acccaaccca attccgtacc atgtcctgca gtgtgaacca catgtcgagc 2040 |
| ccacacaagc cacttacaat ggaaaaatga tcgtcatcag gctctgcctc attgccatcc 2100 |
| cgggcatcca gaggtacaag tgtgcactcg aaggagcatc ttagctgctg cgttacgctt 2160 |
| tgccagcttt ttggaagtgc cactgcctga gggtggggga aggaattgtt caacttgggg 2220 |
| attcctaaca gaccaatgct ttcctctagg gtaacttaga cagtcaccca atgagatacc 2280 |
| actgcaaaga ggagctcaag gcctgatctt tttaactgag gagttggtga gccagataag 2340 |
| agggggaaag atgggcaggg acaagggaga gacaggaagg caggagctga gcaaggacct 2400 |
| gcaaagcaaa ggaagctggg acagagcaca gccaggcaaa gaaccacctt gtgtttgggg 2460 |
| ttacagtgtg gcaaagagag aaggtgagtg aacccccttt tccccagtta ccaatctcaa 2520 |
| tgaaacgctc cacccggcaa gtcatggtga actctttgcg gtgagcaggc ccagactctt 2580 |
| gggtcaccat gtactctggc aaacgccagc ctttttgcac caccagctcc ttgaaagaaa 2640 |
| accataagag agtccaaggc ttagtgagga aagggtcctt agccaatggc cttctaccaa 2700 |
| gagcctccac ctttgagtga gaaggaggac caccctaccc acaagtgcac acgcccctgc 2760 |
| ctcagcccta cttcagagaa gatagcacac atgacaatga agacgaggca cacctgcaga 2820 |
| gcaccgacgg ggttgcactc agactgctga ggagagacag ggggctgcat ctccatggga 2880 |
| gggctcctaa agaaaaaagg gcccaggcca gtgctgagga aaatgcagag gttcctcctg 2940 |
| tcctggtcat ggatcaagtg ccagccacta ccacctccca attcctcacc aggcaggcag 3000 |
| ctatctgtcc aaacatctag cctttctcct ccctccccac accccaccaa gtgaggctct 3060 |
| gctacttccc aggagcactt agatgtggaa atccttcagc aatgtccctg tgatggaggt 3120 |
| aaaggagctg tacacacagt cctggaagta ccttttgggt ctcaaacatg gagcctctaa 3180 |
| gatgtttaaa caatctaaca ttcattgctt ggctatttaa tcatcataaa ttacctccta 3240 |
| gctcaggctt accaaccagc tggtgcaaat ttttcaatta caccagtctg tctttactct 3300 |
| caggcttcct catcctgacc tgcctgagtc caagagctat gagactagaa gccaagaccc 3360 |
| cctccttcct ccaagacgtc aggggactca agatacctgg ttagtacagc agatggaaca 3420 |
| ggggcagcag cttctgcagc aacgacagga gtgtcctcag gcggtgaaga gtctaggaga 3480 |
| gaaaaagaac ttcccgaggg gaagaaggtc ttggattcac acccatgtag aagggaatga 3540 |
| gggtgtgagg tccttctgac ctttccatcc cctgagaact ccttcccccc gatgcagact 3600 |
| cggagagcct cagcaacagc tcttcctcct gggatcctct gctgacccca cactggctca 3660 |
| gaagcactga ggttagggtg ggggctcaga aaagagtctc tttctaccca acaagctgca 3720 |
| acctgaatgt ggaagaggga aagccatctt gctagaaaga gggggcatgc caggaccaac 3780 |
| tttatcagtc tttgcctcca ccccctatgc ccttttctca tgccccagag cagccaccac 3840 |
| ccatcaaagg gggctggcta ttactctgag gcactctaga aacaccctgt tcagcttcag 3900 |
| aggaaattct caggaagggg ccacgagagg gtggccatga gtcaccagac caggcttagg 3960 |
| gagagaagta gatagtgcag aggcctgggt tctcagcggg cttctagtgt gccttggctg 4020 |
| ttcctccctc acctgctgtc ctccagggct ggttccagca tgctcccccc tttgaggtgt 4080 |
| ttgagggcca cctcagctgc cttgtgcttg gctgccttct tgctggggcc ctgacctaag 4140 |
| aaaaggggcg tgggcaatac tggaggtcac cggcaggaca gcagagaaac cagcccccac 4200 |
| agcctatctc ctcatagcca gagggagtga gaagagccct ctgaccaacc tcccctgtca 4260 |
| gaactggaag ggttctacta attgttggac tagccttttt cctagttagc attaacagtc 4320 |
| aacttggcac aacctaggat ctaaaaggat ctgaaaggag agccttaact gaggaattgc 4380 |
| ctacatcagc ctggcctgag ggcatgtctg ggggtgggtg ggggggatgg actgatagtt 4440 |
| aattgatgct gatatgcaca gcccactatt gtggtaccac ccctaggcag gtggtcctga 4500 |
| actgtgtaag aaatctagta gagtataaat aagccagcca gccagcatgg agtatctgcc 4560 |
| attgttcctg ctgtgcttct gtggctgtga agtgagttcc ttagtcagaa gtagcaagaa 4620 |
| tcagattatt ttctttggtc ttgactgata atgtgagttt cttccttagc ttcccttagt 4680 |
| catgaatgta acctgaactg taaacctatc aacccttttc ctcctctaaa ctgcttttag 4740 |
| tttcggagcc ttactacagc aacagaaatg ggccttgaac acctcctctt tcagtcagct 4800 |
| cccgtaatga aactgtgcca ggcaggattt ttgagctcca acactggtgg gttgcggagg 4860 |
| gatgttttgc tctgtctttc tttccccttt tctcttccct cggtaaatag gaaatagaga 4920 |
| caactgaggg tctgaagtca aagctctgct ctgccactta ttacatgtga actggagcct 4980 |
| accatttaat ttctcatact aagttcttcg tgttcaatgt gaggcatgaa ggctagcaga 5040 |
| gtatgtgctc cccctaatac ccacgaggag ttcaatgtct catttactct tcctctgggg 5100 |
| tgataaagta gcagatccga attc 5124 |
| <200> SEQUENCE CHARACTERISTICS: |
| <210> SEQ ID NO 3 |
| <211> LENGTH: 2480 |
| <212> TYPE: DNA |
| <213> ORGANISM: Homo sapiens |
| <400> SEQUENCE: 3 |
| tggcttagtc cgcattaaca gaaatatggt atctggagca tccagatcca aacctgctct 60 |
| aatctacgtg tctaaaggat aagttggttc ctagatgcct tgccttaaag agtggggtgc 120 |
| ttccccttga gcaaaaaaga ctatggagtg ggccaggtgc ggtggctcac gcctgtaatc 180 |
| ccaacacttt gggatgccaa ggtgggtgga ttgcttgagg tcaggagttc aagaccagcc 240 |
| tggccaacat ggtgaaaccc catctctact ccccctacaa aaattagccg ggcatagtgg 300 |
| tgtgtgcctg taatcccagc tacttgggag gctgaggcag gagaatcgcc tgaacccggg 360 |
| aggcagaggg gttgcggtga gccgagatcg caccactgca ctccagcctg ggtgacagag 420 |
| ccagactcca tcgtaaaaaa aaaaaaaaaa aagctggagt gtaagaatga actctctctt 480 |
| caaaaatatg aagagctttt acacatgaaa gagatgagat gttctgttga gacttaatgg 540 |
| agcaaactaa gaccccgagt agaagtcaca ggaagcccaa agccccctcg gcacaaggat 600 |
| aaatattgtc cgaagactgt ctgaggagat ggtaagttcc ccgactgggg cacatgaggc 660 |
| aatcacagct aacgttttga ggatacctgt tctatgcaag ctgttttatt gaatggatta 720 |
| caaaaagtat gttaagtaat gcacacccta ggaaggcgtt ccttgtaaaa tgggcccatg 780 |
| tttcaggtga ggaaactgaa gctcagagcg ggagacagct tgctcaagca gaggccaagg 840 |
| cctttacgtc atggctcttc ctcagcacaa gctagagacc cacactggtg gggatgctcc 900 |
| aaaagggact cgaggattag aaggaacctg caccatatgg ctttgaaggc ctctccctga 960 |
| ggattagtga ttctgatttt tcttaagaca ggattctagg ctactctata tgagagccag 1020 |
| ggccaggcag aggctgtgga gaccagagtc ggggtcagaa tgatagtcca gccccacagt 1080 |
| agcctgctcc tgtccttctg ggactccctg gagactggac cctagcacct ccactcagcc 1140 |
| tcaccctcct cacttcctct gcagaggtct attctaggaa aaggaaatgg ccggagctgg 1200 |
| caatgaggag ctgtgaccac agggtggcag cagtgcttca gctgtggaga gtgactatag 1260 |
| gaagggcgat gaggcaggtc ggaagcagtg ctctctagtg aggatggggg tctgtctgca 1320 |
| aagattggaa gccacactca ttcagtgggc tccaaaatcc tgtagcctcc ctctatatct 1380 |
| taataatttt ttttttttga gacagagttt ctctttttgc caaggctgga gtgtagtggt 1440 |
| gccatctcag ctcactgcaa cctctgcctc cccgattcaa gcgattctcc tgcctcagcc 1500 |
| tcctgagtag ctgggattac aggtgcctac caccacgccc agctaatttt tgtattttta 1560 |
| gtagacaggg gtttcaccat gttggccagg ctggtctcga actcctgccc tcaggtgatc 1620 |
| cacccgcctt ggtctcccaa agtgctgggg ttacaggtgt gaggcactgc acccggcaaa 1680 |
| aaaaaaatgg tttttaatta aaaaaaaaaa gatacaggct gggcatggtg gttgacgcct 1740 |
| gtagtcccag ctacttggga ggctgaggca ggagaatcac ttgaacccag gagccagagg 1800 |
| ttgcagtgag ccgagatcgc gccactgcac tccagcctgg gcaaaaagag cgaaactcca 1860 |
| tctcaaaaag aaaaaaagtt aaattctctc catcatcatg aagttgaata tattttttct 1920 |
| atccacaggc aaatctgagt agcctccaag aggcacacaa gcagaggatg ggctgtgttg 1980 |
| ccctgactgc cagccccagg cacagaggac caggcctggt catcctcaca gactctgacc 2040 |
| ctggctcttc ccactcctct tccactccag gacatcctac ttaacccctc ctgacatgag 2100 |
| tttcttgtgc tttagtctac aggttaggaa agaggggaag tgataaacaa gctctccaac 2160 |
| ctgttgaggg attaggggtt cgtctaaggc tccccagggc ctggctctga caaagcgtct 2220 |
| gcaactaatg atgcttcttg agctctggag acaggattta tgcatctaat aaagtctgta 2280 |
| actccaggct taggggccgg gggcaggagg ctgagagcat gaagtcctgg gggcgccatg 2340 |
| ggaggagatc ccaggtggct cctaatgagc cctgcatttc atttgcctgc tctagattcc 2400 |
| cctaaggcta ctgtgaggct gggggtgggg gaacagcagg tataagaggt tggggtggct 2460 |
| gtaggagggt aggtggcagc 2480 |
| <200> SEQUENCE CHARACTERISTICS: |
| <210> SEQ ID NO 4 |
| <211> LENGTH: 396 |
| <212> TYPE: DNA |
| <213> ORGANISM: Mouse |
| <220> FEATURE: |
| <221> NAME/KEY: CDS |
| <222> LOCATION: (7)..(393) |
| <400> SEQUENCE: 4 |
| gtatct atg aat ata gct ctg ttc tat atc ctt ttg aca tgt agc aat 48 |
| Met Asn Ile Ala Leu Phe Tyr Ile Leu Leu Thr Cys Ser Asn |
| 1 5 10 |
| acc tct cca tcc tca agg aac tca acc cag tct ggg tct ccc cag gct 96 |
| Thr Ser Pro Ser Ser Arg Asn Ser Thr Gln Ser Gly Ser Pro Gln Ala |
| 15 20 25 30 |
| cca gtg gta gac tct gac agg tgg gag gat aca gtg ctc tgg gct gtt 144 |
| Pro Val Val Asp Ser Asp Arg Trp Glu Asp Thr Val Leu Trp Ala Val |
| 35 40 45 |
| ttg tta caa aag tgt ctt ctg tcc ttt ccc tcc tcc caa ttc agc atg 192 |
| Leu Leu Gln Lys Cys Leu Leu Ser Phe Pro Ser Ser Gln Phe Ser Met |
| 50 55 60 |
| acc cct gtg agc agg ctc tca caa tct cct ggg gca ggg ctg agg cag 240 |
| Thr Pro Val Ser Arg Leu Ser Gln Ser Pro Gly Ala Gly Leu Arg Gln |
| 65 70 75 |
| ggg ctt tca gct ctt ctc cat aac tat ccc ttc ttc ctt ccc cca tgc 288 |
| Gly Leu Ser Ala Leu Leu His Asn Tyr Pro Phe Phe Leu Pro Pro Cys |
| 80 85 90 |
| cat tta gca gtt atc acc cag cct tgc ctt ctc cct cca tcc ctt gcc 336 |
| His Leu Ala Val Ile Thr Gln Pro Cys Leu Leu Pro Pro Ser Leu Ala |
| 95 100 105 110 |
| ctg aca tat act gtg cct tat tta tgc tgc aaa tat aac att aaa cta 384 |
| Leu Thr Tyr Thr Val Pro Tyr Leu Cys Cys Lys Tyr Asn Ile Lys Leu |
| 115 120 125 |
| tca aga gaa tga 396 |
| Ser Arg Glu |
| <200> SEQUENCE CHARACTERISTICS: |
| <210> SEQ ID NO 5 |
| <211> LENGTH: 129 |
| <212> TYPE: PRT |
| <213> ORGANISM: Mouse |
| <400> SEQUENCE: 5 |
| Met Asn Ile Ala Leu Phe Tyr Ile Leu Leu Thr Cys Ser Asn Thr Ser |
| 1 5 10 15 |
| Pro Ser Ser Arg Asn Ser Thr Gln Ser Gly Ser Pro Gln Ala Pro Val |
| 20 25 30 |
| Val Asp Ser Asp Arg Trp Glu Asp Thr Val Leu Trp Ala Val Leu Leu |
| 35 40 45 |
| Gln Lys Cys Leu Leu Ser Phe Pro Ser Ser Gln Phe Ser Met Thr Pro |
| 50 55 60 |
| Val Ser Arg Leu Ser Gln Ser Pro Gly Ala Gly Leu Arg Gln Gly Leu |
| 65 70 75 80 |
| Ser Ala Leu Leu His Asn Tyr Pro Phe Phe Leu Pro Pro Cys His Leu |
| 85 90 95 |
| Ala Val Ile Thr Gln Pro Cys Leu Leu Pro Pro Ser Leu Ala Leu Thr |
| 100 105 110 |
| Tyr Thr Val Pro Tyr Leu Cys Cys Lys Tyr Asn Ile Lys Leu Ser Arg |
| 115 120 125 |
| Glu |
| <200> SEQUENCE CHARACTERISTICS: |
| <210> SEQ ID NO 6 |
| <211> LENGTH: 1920 |
| <212> TYPE: DNA |
| <213> ORGANISM: Mouse |
| <400> SEQUENCE: 6 |
| tacagagtct caggcttaga aaaccagaca agctgggcac tgtgacacac actttaatcc 60 |
| cagcacttgg gaggcagagg catcctaatc tacatagtaa attctagacc agccaaggct 120 |
| aaatggtgac acctgtttac acacacacac acacacacac acacacacac acacacacac 180 |
| acacacacac acacacagtg gagacaggaa aagagagagg tggggagaga aaccgagaag 240 |
| acccacaaca aaagcagcag tgaaatattt caactataat gtatgacaag ctctacagga 300 |
| aatcttgaga caaaccttac agaaaggttc acccaagcct ggatgcattg tattctagaa 360 |
| catcagaaac ctgatctaga aggtctccct ctgcagtaga gcacctgcct accatacaca 420 |
| aggctcttca tttggacttg attcccaaga aagaaatttt aaaatgccac ttatccgcaa 480 |
| acctaaaatg taagttggtt ccaataaagt acttccctgt tgagcaaaga agatgtggat 540 |
| acatggtatt tctccaaata tgtgaggggc ttttacatat gactgagaga taacctgaga 600 |
| ctccattgaa caagctagga cctctggtag aggccaccaa gaagctaaag cccactcagc 660 |
| actctctgga gatggtaagt tccccaggac tggagtggga ggacagcaaa gggaatcaca 720 |
| gttgacattt tgaaaacacc gggtctgtgc tttcctacaa aatgcatccc aaatgtttct 780 |
| cctagcaagt aattcatttt actgttccca tatgtaagtg aggaaaaaaa gagtgtgagc 840 |
| agcttgcttt gctcatgggg tagaaccccg acagtccttc ttctgtttag gctagagaca 900 |
| tggtactctg acacctggat ttgcaagtga ggttaggact agctccttta aaggaccctt 960 |
| ccctgaactg gagtgattgt ctgtccctaa agcagaaccc tagtcgccag ctccagtagt 1020 |
| atattagaac cagaaccagg cagagcccat gctgaccaga cggaactgga aaaatgtcac 1080 |
| aattctgggc cccaaagaac taggtcctca agtcctagac aaaatgtatg gaaagggaaa 1140 |
| tggctggacg tggcagtgaa gagtagtggc cacaaggtgg cagcagagtt tcagctgtgg 1200 |
| aggcccaatc cccagttctc ttgcaaagat gggcctgtcc acaaaattta caggccacct 1260 |
| ctactcagta aggctccaaa aagagtctcc tatctctcac ttaactattc acaggtaaat 1320 |
| cttaaagggt agtgaaccca catttaacct gactagaagc agtggggatt gaaatggggc 1380 |
| tgtggtcctg atcacccatt ccaggcagga gtagggacca agctggttca ccctagcctg 1440 |
| cacttaacac tagttccttc ccatccagga cataatgccc aattctgaca ggagtttctc 1500 |
| cagtcaggaa caagaggtga tcaattgaag cttctccaat ctgttgaagg attggaggtt 1560 |
| cttctaaggt tcccccaggg tctaactctg acaaactgtc tgcaattaat gatgcttcct 1620 |
| gagctccgga gacaagattt atgcatctaa taaagtctat ataactccag gcttaggctg 1680 |
| ggggggagga agctaagagc agagagtccc caggggagta cgggaggggg ggtcccaggt 1740 |
| ggctcttaat agagccatgc atttccattg cttgtctaga tttcccccca ggctgccggt 1800 |
| gaggtggggg tagggacatc aggtataaga agaccgtggg cactcaggag gcagatggca 1860 |
| gcatggactc caagtgggct gctctgctgc tgctgctact gctgctgctg aattggggcc 1920 |
| <200> SEQUENCE CHARACTERISTICS: |
| <210> SEQ ID NO 7 |
| <211> LENGTH: 411 |
| <212> TYPE: DNA |
| <213> ORGANISM: Mouse |
| <400> SEQUENCE: 7 |
| tccaggacat aatgcccaat tctgacagga gtttctccag tcaggaacaa gaggtgatca 60 |
| attgaagctt ctccaatctg ttgaaggatt ggaggttctt ctaaggttcc cccagggtct 120 |
| aactctgaca aactgtctgc aattaatgat gcttcctgag ctccggagac aagatttatg 180 |
| catctaataa agtctatata actccaggct taggctgggg gggaggaagc taagagcaga 240 |
| gagtccccag gggagtacgg gagggggggt cccaggtggc tcttaataga gccatgcatt 300 |
| tccattgctt gtctagattt ccccccaggc tgccggtgag gtgggggtag ggacatcagg 360 |
| tataagaaga ccgtgggcac tcaggaggca gatggcagca tggactccaa g 411 |
| <200> SEQUENCE CHARACTERISTICS: |
| <210> SEQ ID NO 8 |
| <211> LENGTH: 410 |
| <212> TYPE: DNA |
| <213> ORGANISM: Rat |
| <400> SEQUENCE: 8 |
| tccaggacta taatgctcaa tcctgacagg agtttcttca gtcaggaaca agaggtgatc 60 |
| aattgaagct tctccaacct gttgaaggat tggaggttct tgtaagactc ctccagggcc 120 |
| tagctctgac aaactgtctg caattaataa tgcttcctga gctctggaga caagatttat 180 |
| gcatctaata aagtctataa ctccaggctc atgctggggg tagagaactg agagcagaaa 240 |
| gtctcccagg gcggtatggg aggggggtcc caggtggctc ttaatagagc catgcatttc 300 |
| cattgcctgt ctagatttcc cccaggctgc tgatgaggtg ggggtagggg acatcaggta 360 |
| taagaagccc gtgtgccacg gaggaggcag atggcagcat ggattccaag 410 |
| <200> SEQUENCE CHARACTERISTICS: |
| <210> SEQ ID NO 9 |
| <211> LENGTH: 411 |
| <212> TYPE: DNA |
| <213> ORGANISM: Homo sapiens |
| <400> SEQUENCE: 9 |
| taacccctcc tgacatgagt ttcttgtgct ttagtctaca ggttaggaaa gaggggaagt 60 |
| gataaacaag ctctccaacc tgttgaggga ttaggggttc gtctaaggct ccccagggcc 120 |
| tggctctgac aaagcgtctg caactaatga tgcttcttga gctctggaga caggatttat 180 |
| gcatctaata aagtctgtaa ctccaggctt aggggccggg ggcaggaggc tgagagcatg 240 |
| aagtcctggg ggcgccatgg gaggagatcc caggtggctc ctaatgagcc ctgcatttca 300 |
| tttgcctgct ctagattccc ctaaggctac tgtgaggctg ggggtggggg aacagcaggt 360 |
| ataagaggtt ggggtggctg taggagggta ggtggcagca tggattctag g 411 |
| <200> SEQUENCE CHARACTERISTICS: |
| <210> SEQ ID NO 10 |
| <211> LENGTH: 417 |
| <212> TYPE: DNA |
| <213> ORGANISM: Artificial Sequence |
| <220> FEATURE: |
| <223> OTHER INFORMATION: Description of Artificial Sequence Consensus |
| sequence for NpFF promotor region |
| <400> SEQUENCE: 10 |
| tccaggacna taatgcncaa ttctgacagg agtttctnca ngtcaggaac aagaggtgat 60 |
| caattgaagc ttctccaacc tgttgaagga ttggaggttc ttctaaggct ccnccagggc 120 |
| ctagctctga caaactgtct gcaattaatg atgcttcctg agctctggag acaagattta 180 |
| tgcatctaat aaagtcnnta taactccagg cttanggctn gggggnagga agctgagagc 240 |
| agaaagtcnc cnggggnggn cngggggggg ggtcccaggt ggctcttaat agagccatgc 300 |
| atttccattg cctgtctaga tttcccccca ggctgctgnt gaggtggggg taggggnaca 360 |
| tcaggtataa gaagnccgtg tggcactnag gaggcagatg gcagcatgga ttccaag 417 |
| <200> SEQUENCE CHARACTERISTICS: |
| <210> SEQ ID NO 11 |
| <211> LENGTH: 18 |
| <212> TYPE: DNA |
| <213> ORGANISM: Artificial Sequence |
| <220> FEATURE: |
| <223> OTHER INFORMATION: Description of Artificial Sequence antisense |
| primer for mouse |
| <400> SEQUENCE: 11 |
| tggagtccat gctgccat 18 |
| <200> SEQUENCE CHARACTERISTICS: |
| <210> SEQ ID NO 12 |
| <211> LENGTH: 26 |
| <212> TYPE: DNA |
| <213> ORGANISM: Artificial Sequence |
| <220> FEATURE: |
| <223> OTHER INFORMATION: Description of Artificial Sequencesense |
| primer |
| for 325 bp mouse promoter |
| <400> SEQUENCE: 12 |
| gtgctagcaa tctgttgaag gattgg 26 |
| <200> SEQUENCE CHARACTERISTICS: |
| <210> SEQ ID NO 13 |
| <211> LENGTH: 26 |
| <212> TYPE: DNA |
| <213> ORGANISM: Artificial Sequence |
| <220> FEATURE: |
| <223> OTHER INFORMATION: Description of Artificial Sequencesense |
| primer |
| for 579bp mouse promoter |
| <400> SEQUENCE: 13 |
| gtgctagcag tctcctatct ctcact 26 |
| <200> SEQUENCE CHARACTERISTICS: |
| <210> SEQ ID NO 14 |
| <211> LENGTH: 26 |
| <212> TYPE: DNA |
| <213> ORGANISM: Artificial Sequence |
| <220> FEATURE: |
| <223> OTHER INFORMATION: Description of Artificial Sequencesense |
| primer |
| for 805bp mouse promoter |
| <400> SEQUENCE: 14 |
| gtgctagcag acggaactgg aaaaat 26 |
| <200> SEQUENCE CHARACTERISTICS: |
| <210> SEQ ID NO 15 |
| <211> LENGTH: 26 |
| <212> TYPE: DNA |
| <213> ORGANISM: Artificial Sequence |
| <220> FEATURE: |
| <223> OTHER INFORMATION: Description of Artificial Sequencesense |
| primer |
| for 1085bp mouse promoter |
| <400> SEQUENCE: 15 |
| gtgctagctc tcctagcaag taattc 26 |
| <200> SEQUENCE CHARACTERISTICS: |
| <210> SEQ ID NO 16 |
| <211> LENGTH: 26 |
| <212> TYPE: DNA |
| <213> ORGANISM: Artificial Sequence |
| <220> FEATURE: |
| <223> OTHER INFORMATION: Description of Artificial Sequencesense |
| primer |
| for 1289bp mouse promoter |
| <400> SEQUENCE: 16 |
| gtgctagcta catatgactg agagat 26 |
| <200> SEQUENCE CHARACTERISTICS: |
| <210> SEQ ID NO 17 |
| <211> LENGTH: 26 |
| <212> TYPE: DNA |
| <213> ORGANISM: Artificial Sequence |
| <220> FEATURE: |
| <223> OTHER INFORMATION: Description of Artificial Sequencesense |
| primer |
| for1527bp mouse promoter |
| <400> SEQUENCE: 17 |
| gtgctagcag cctggatgca ttgtat 26 |
| <200> SEQUENCE CHARACTERISTICS: |
| <210> SEQ ID NO 18 |
| <211> LENGTH: 26 |
| <212> TYPE: DNA |
| <213> ORGANISM: Artificial Sequence |
| <220> FEATURE: |
| <223> OTHER INFORMATION: Description of Artificial Sequencesense |
| primer |
| for 1861bp mouse promoter |
| <400> SEQUENCE: 18 |
| gtgctagcac agagtctcag gcttag 26 |
| <200> SEQUENCE CHARACTERISTICS: |
| <210> SEQ ID NO 19 |
| <211> LENGTH: 23 |
| <212> TYPE: DNA |
| <213> ORGANISM: Artificial Sequence |
| <220> FEATURE: |
| <223> OTHER INFORMATION: Description of Artificial Sequence antisense |
| primer for human |
| <400> SEQUENCE: 19 |
| gctgccacca cctaccctcc tac 23 |
| <200> SEQUENCE CHARACTERISTICS: |
| <210> SEQ ID NO 20 |
| <211> LENGTH: 21 |
| <212> TYPE: DNA |
| <213> ORGANISM: Artificial Sequence |
| <220> FEATURE: |
| <223> OTHER INFORMATION: Description of Artificial Sequence antisense |
| primer for rat |
| <400> SEQUENCE: 20 |
| caccccagct ccctgcctct t 21 |
| <200> SEQUENCE CHARACTERISTICS: |
| <210> SEQ ID NO 21 |
| <211> LENGTH: 26 |
| <212> TYPE: DNA |
| <213> ORGANISM: Artificial Sequence |
| <220> FEATURE: |
| <223> OTHER INFORMATION: Description of Artificial Sequence antisense |
| primer for nested human |
| <400> SEQUENCE: 21 |
| gtggatccat ctagagcagg caaatg 26 |
| <200> SEQUENCE CHARACTERISTICS: |
| <210> SEQ ID NO 22 |
| <211> LENGTH: 21 |
| <212> TYPE: DNA |
| <213> ORGANISM: Artificial Sequence |
| <220> FEATURE: |
| <223> OTHER INFORMATION: Description of Artificial Sequence antisense |
| primer for nested rat |
| <400> SEQUENCE: 22 |
| cgtggcccca gttcctcagc a 21 |
Panula, Pertti Aarre Juhani, Brandt, Annika, Westerlund, Johanna
| Patent | Priority | Assignee | Title |
| 7341847, | Apr 02 2003 | Agency for Science, Technology and Research | Promoter construct for gene expression in neuronal cells |
| 9724430, | Sep 28 2007 | Intrexon Corporation | Therapeutic gene-switch constructs and bioreactors for the expression of biotherapeutic molecules, and uses thereof |
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